Recombinant Perisphaeria ruficornis Pyrokinin-5

What is Perisphaeria ruficornis Pyrokinin-5 and what is its amino acid sequence?

Perisphaeria ruficornis Pyrokinin-5 is a neuropeptide isolated from the cockroach species Perisphaeria ruficornis. It belongs to the pyrokinin/PBAN (pheromone biosynthesis activating neuropeptide) family of peptides characterized by a conserved FXPRLamide C-terminal sequence. The complete amino acid sequence of this peptide is SGETSGEGNGMWFGPRL, with the C-terminal pentapeptide (FGPRL) serving as the active core required for its biological functions . The peptide is also known by alternative names including FXPRL-amide and PerRu-Capa-PK .

What expression systems are used to produce recombinant Perisphaeria ruficornis Pyrokinin-5?

Recombinant Perisphaeria ruficornis Pyrokinin-5 can be produced using several expression systems:

• E. coli: The most commonly used system for producing this recombinant peptide with >85% purity as determined by SDS-PAGE

• Yeast expression systems

• Baculovirus expression systems

• Mammalian cell expression systems

The choice of expression system depends on research requirements, including post-translational modifications, yield, and downstream applications. E. coli remains the predominant system due to its cost-effectiveness and relatively high protein yields.

How should Recombinant Perisphaeria ruficornis Pyrokinin-5 be reconstituted and stored for optimal stability?

For optimal reconstitution and storage:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (typically 50% is recommended) for long-term storage stability

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

• For extended storage, keep at -20°C or preferably -80°C

• Avoid repeated freeze-thaw cycles, as these significantly decrease protein activity

The shelf life is approximately 6 months for liquid formulations at -20°C/-80°C and 12 months for lyophilized formulations at the same temperatures .

What are the known physiological functions of pyrokinin/PBAN peptides in insects?

Pyrokinin/PBAN peptides serve diverse physiological functions across different insect species:

• Stimulation of pheromone biosynthesis in female moths, critical for reproductive behavior

• Induction of muscle contractions, particularly in the hindgut of insects

• Regulation of embryonic diapause in Bombyx mori (silkworm)

• Stimulation of melanization processes in certain larval moth species

• Acceleration of puparium formation in dipteran insects

• Involvement in neuroendocrine signaling via the abdominal perisympathetic organs (PSOs)

• Potential roles in metabolic stress responses

The pentapeptide C-terminal sequence (FXPRLamide) serves as the active core required for these diverse physiological functions, binding to specific G protein-coupled receptors to initiate downstream signaling events .

How can Perisphaeria ruficornis Pyrokinin-5 be used in phylogenetic studies of insects?

Perisphaeria ruficornis Pyrokinin-5, along with other CAPA peptides, has proven valuable for phylogenetic analyses of insect taxa for several reasons:

• The sequences of these neuropeptides contain phylogenetically informative substitutions that can complement molecular and morphological data

• These peptides can be directly analyzed from single specimens using mass spectrometry, requiring minimal sample preparation

• The conserved nature of certain regions (like PVK-2) and the variable nature of others provide a balanced dataset for evolutionary studies

• When combined with other neuropeptide data (such as adipokinetic hormones and sulfakinins), they produce robust phylogenetic trees with improved bootstrap values

Research has demonstrated that cladograms derived from CAPA peptide sequences show topologies generally consistent with recent molecular and morphological phylogenetic analyses, including the placement of termites within cockroaches .

What is the relationship between Pyrokinin-5 and other CAPA peptides in the insect neuroendocrine system?

Pyrokinin-5 belongs to a broader family of CAPA peptides that form a crucial component of the insect neuroendocrine system:

• CAPA genes typically encode up to four peptides belonging to two groups:

  • CAPA-periviscerokinins (PVKs)

  • CAPA-pyrokinins (PKs, which include Pyrokinin-5)

• These peptides bind to different receptor types, indicating distinct signaling pathways and functions

• CAPA peptides are expressed in:

  • A few interneurons in the central nervous system

  • The neuroendocrine system of the abdominal ventral nerve cord

  • Abdominal perisympathetic organs (PSOs), from which they are released into the hemolymph

• Most cockroach species express three different PVKs and one PK, though some species (like Cryptocercus and certain blattellid cockroaches) express only two PVKs

• Some Madagascan Blaberidae and the Table Mountain cockroach (Aptera fusca) express a fourth PVK (PVK-4) that appears to result from internal gene duplication events

What analytical techniques are recommended for studying Perisphaeria ruficornis Pyrokinin-5 in biological samples?

For studying Perisphaeria ruficornis Pyrokinin-5 in biological samples, the following analytical approaches are recommended:

• Mass Spectrometry:

  • Tandem mass spectrometry (MS/MS) is particularly effective for obtaining sequence data directly from perisympathetic organs of single specimens

  • Direct mass spectrometric screening of abdominal PSO preparations allows for rapid characterization of CAPA peptides

  • MALDI-TOF MS is suitable for initial peptide identification

• Immunocytochemical Techniques:

  • Polyclonal antisera against the C-terminal ending can reveal the location of cell bodies and axons in the central nervous system

  • Useful for mapping the distribution of pyrokinin/PBAN-like peptides in larval and adult insect nervous systems

• Bioassays:

  • Pheromonotropic activity can be assessed using moth models such as Helicoverpa zea and Helicoverpa armigera

  • Muscle contraction assays help evaluate myotropic effects

  • Puparium formation assays in dipterans can assess developmental effects

These techniques can be used complementarily to provide a comprehensive understanding of the peptide's expression, distribution, and biological activity.

How can researchers effectively design functional assays to study the biological activity of Pyrokinin-5?

Designing effective functional assays for Pyrokinin-5 requires careful consideration of its known biological activities and experimental controls:

• Receptor Binding Assays:

  • Express G protein-coupled receptors (GPCRs) in heterologous cell systems

  • Measure calcium mobilization or cAMP production following peptide application

  • Include dose-response curves (typically 10⁻¹² to 10⁻⁶ M peptide concentrations)

  • Use structure-activity relationship studies with modified peptides (especially alterations to the FXPRLamide core)

• Muscle Contraction Assays:

  • Isolate insect hindgut tissues and mount in organ baths with physiological saline

  • Record spontaneous and peptide-induced contractions using force transducers

  • Compare native peptide activity with synthetic analogs

  • Include positive controls (other known myotropic peptides) and vehicle controls

• Pheromone Biosynthesis Assays:

  • Utilize moth pheromone gland preparations

  • Measure fatty acid incorporation or pheromone component production

  • Perform time-course experiments to determine optimal incubation periods

  • Include comparative analyses with other PBAN peptides

• Controls and Validation:

  • Include scrambled peptide sequences as negative controls

  • Verify peptide integrity before assays using HPLC or mass spectrometry

  • Conduct dose-dependent studies to establish EC₅₀ values

  • Perform receptor antagonist studies to confirm specificity of response

What are the key considerations when comparing Pyrokinin-5 activity across different insect species?

When comparing Pyrokinin-5 activity across different insect species, researchers should consider:

• Evolutionary Relationships:

  • Phylogenetic distance between species affects receptor-ligand interactions

  • Consider the evolutionary history of CAPA/pyrokinin signaling systems

  • Interpret cross-species activities in light of evolutionary conservation or divergence

• Receptor Homology:

  • Sequence similarity of pyrokinin receptors across species

  • Potential differences in receptor subtypes and their distribution

  • Possibility of receptor promiscuity (one receptor responding to multiple related peptides)

• Methodological Standardization:

  • Use consistent assay conditions across species comparisons

  • Standardize peptide concentrations and application methods

  • Account for species-specific differences in experimental tissues (e.g., size, innervation)

• Physiological Context:

  • Consider life-stage specific differences in peptide activity

  • Account for environmental factors that might affect receptor sensitivity

  • Evaluate potential interactions with other neuroendocrine systems

• Structure-Activity Relationships:

  • Compare the effects of sequence variations in the core pentapeptide region

  • Evaluate the importance of N-terminal extensions for species-specific activities

  • Consider potential differences in post-translational modifications

How do CAPA-pyrokinins interact with their G protein-coupled receptors at the molecular level?

The molecular interaction between CAPA-pyrokinins and their G protein-coupled receptors involves:

• Binding Determinants:

  • The C-terminal FXPRLamide motif is essential for receptor recognition and activation

  • The amidated C-terminus forms hydrogen bonds with conserved receptor residues

  • The arginine residue in the core sequence provides critical ionic interactions

  • The proline creates a characteristic bend in the peptide backbone that facilitates receptor binding

• Receptor Activation Mechanism:

  • Binding induces conformational changes in transmembrane domains

  • This conformational shift allows interaction with intracellular G proteins

  • Primarily couples to Gq/11 proteins, activating phospholipase C signaling pathways

  • Leads to increased intracellular calcium and protein kinase C activation

• Receptor Subtypes:

  • Different receptors exist for PVKs versus PKs

  • Species-specific variations in receptor structure affect binding affinity

  • Some evidence suggests receptor desensitization following prolonged exposure

• N-terminal Region Effects:

  • While the C-terminal pentapeptide is critical for binding, the N-terminal region of Pyrokinin-5 (SGETSGEGNGMW) can influence:

    • Receptor subtype selectivity

    • Binding kinetics

    • Signaling efficacy

    • Resistance to enzymatic degradation

Understanding these molecular interactions provides insights for developing receptor agonists or antagonists with potential applications in insect control strategies.

What is the relationship between peptide sequence conservation and functional evolution in the pyrokinin family?

The relationship between sequence conservation and functional evolution in the pyrokinin family reveals several important patterns:

• Sequence Conservation Patterns:

  • The C-terminal FXPRLamide motif shows strong conservation across diverse insect taxa, reflecting its critical role in receptor binding

  • PVK-2 sequences are highly conserved and contain few phylogenetically informative substitutions

  • N-terminal regions show greater variability, suggesting functional adaptation

  • Sequence table comparison across species (from search result ):

• Functional Diversification:

  • Despite sequence conservation, pyrokinins have evolved diverse functions:

    • Pheromone biosynthesis regulation

    • Myotropic activity

    • Melanization control

    • Diapause regulation

  • This functional diversity likely arose through:

    • Receptor duplication and diversification

    • Changes in peptide expression patterns

    • Co-evolution with downstream signaling pathways

• Evolutionary Significance:

  • Internal gene duplications have led to additional peptide variants (e.g., PVK-4 in some species)

  • Pyrokinin sequence conservation provides strong phylogenetic signals that complement molecular data

  • The presence of similar peptides across distant insect orders suggests ancient origins and fundamental physiological roles

• Structure-Function Relationships:

  • Even single amino acid substitutions in the core region can significantly alter receptor binding profiles

  • The variable N-terminal regions may confer target tissue specificity

  • Post-translational modifications further diversify functional properties

This evolutionary pattern suggests that while the core signaling mechanism is ancient and conserved, the pyrokinin system has been co-opted for diverse physiological functions through subtle sequence variations and changes in expression patterns.

What are the current limitations in studying Pyrokinin-5 signaling pathways and potential strategies to overcome them?

Current limitations in studying Pyrokinin-5 signaling pathways include:

• Receptor Characterization Challenges:

  • Limited structural information on pyrokinin receptors

  • Difficulty in expressing functional insect GPCRs in heterologous systems

  • Multiple receptor subtypes with overlapping ligand specificities

  Potential Solutions:

  • Cryo-EM or X-ray crystallography of receptor-ligand complexes

  • Development of receptor-specific antibodies for localization studies

  • CRISPR-based receptor knockout models to identify specific functions

• Signaling Cascade Complexity:

  • Cross-talk between pyrokinin signaling and other neuropeptide pathways

  • Tissue-specific differences in downstream effectors

  • Temporal dynamics of signaling not well characterized

  Potential Solutions:

  • Phosphoproteomic approaches to map signaling networks

  • Real-time imaging using fluorescent biosensors for calcium and other second messengers

  • Single-cell transcriptomics to identify cell-specific response patterns

• Physiological Context Limitations:

  • In vitro studies may not recapitulate in vivo complexity

  • Species-specific differences complicate extrapolation

  • Developmental stage-specific effects are poorly understood

  Potential Solutions:

  • Development of ex vivo organ culture systems

  • Optogenetic approaches for temporal control of peptide release

  • Comparative studies across developmental stages

• Technical Limitations:

  • Limited availability of specific antibodies and antagonists

  • Challenges in measuring endogenous peptide release

  • Difficulty in distinguishing direct vs. indirect effects

  Potential Solutions:

  • Development of specific receptor antagonists and agonists

  • Application of microdialysis techniques for measuring peptide release

  • Combinatorial approaches using genetic and pharmacological tools

• Integration with Other Physiological Systems:

  • Understanding how Pyrokinin-5 signaling integrates with broader physiological processes

  • Identifying environmental factors that modulate signaling effectiveness

  Potential Solutions:

  • Systems biology approaches to model network interactions

  • Multi-omics studies combining transcriptomics, proteomics, and metabolomics

  • Ecological studies examining signaling under natural conditions

Addressing these limitations will require interdisciplinary approaches combining molecular biology, structural biology, systems neuroscience, and evolutionary biology perspectives.

What quality control parameters should be assessed when working with recombinant Pyrokinin-5?

Researchers should evaluate the following quality control parameters when working with recombinant Pyrokinin-5:

• Purity Assessment:

  • SDS-PAGE analysis (should show >85% purity)

  • HPLC profiles to detect impurities

  • Mass spectrometry to confirm molecular weight and potential contaminants

  • Endotoxin testing if the peptide was produced in bacterial systems

• Sequence Verification:

  • Mass spectrometry (MS/MS) to confirm the correct amino acid sequence

  • N-terminal sequencing for additional validation

  • Assessment of potential post-translational modifications

  • Verification of C-terminal amidation, which is critical for biological activity

• Structural Integrity:

  • Circular dichroism to assess secondary structure

  • NMR for detailed structural characterization

  • Stability testing under various storage conditions

• Functional Validation:

  • Receptor binding assays

  • Calcium mobilization assays

  • Comparison with synthetic peptide standards

  • Bioactivity testing in appropriate model systems

• Batch Consistency:

  • Lot-to-lot variation assessment

  • Stability under reconstitution conditions

  • Reproducibility of functional responses

  • Shelf-life determination under recommended storage conditions

Thorough quality control ensures experimental reproducibility and reliable research outcomes when working with this neuropeptide.

What are optimal experimental designs for studying the tissue-specific effects of Pyrokinin-5?

Optimal experimental designs for studying tissue-specific effects of Pyrokinin-5 include:

• Ex Vivo Tissue Preparations:

  • Isolated hindgut contractility assays

  • Pheromone gland incubations from lepidopteran species

  • Neuronal preparations to measure electrophysiological responses

  • Tissue-specific culture systems with controlled exposure parameters

• Receptor Localization Approaches:

  • Immunohistochemistry using anti-receptor antibodies

  • In situ hybridization to detect receptor mRNA expression

  • Receptor-reporter gene constructs in transgenic models

  • Single-cell RNA sequencing to identify receptor-expressing cell populations

• Functional Genomics:

  • CRISPR-Cas9 receptor knockout in specific tissues

  • RNAi-mediated receptor knockdown

  • Tissue-specific overexpression of receptors

  • Conditional expression systems for temporal control

• Pharmacological Approaches:

  • Dose-response relationships in different tissues

  • Competitive binding with receptor antagonists

  • Structure-activity studies using modified peptides

  • Comparison with other CAPA peptides to determine specificity

• Imaging Techniques:

  • Calcium imaging to visualize real-time responses

  • FRET-based approaches to monitor receptor activation

  • Confocal microscopy for subcellular localization

  • Whole-animal imaging using fluorescent reporters

• Experimental Controls:

  • Vehicle controls (appropriate solvents without peptide)

  • Negative controls using inactive peptide analogs

  • Positive controls with known tissue-specific activators

  • Internal controls with housekeeping genes or constitutively active pathways

These comprehensive experimental approaches allow for detailed characterization of tissue-specific responses to Pyrokinin-5, providing insights into its diverse physiological functions.

How can researchers effectively combine molecular, biochemical, and physiological approaches to comprehensively study Pyrokinin-5?

An integrated research strategy for Pyrokinin-5 should combine multiple methodological approaches:

• Sequential Investigation Framework:

  • Begin with molecular characterization (sequence, structure)

  • Proceed to biochemical analyses (receptor binding, signaling)

  • Extend to cellular responses (calcium dynamics, gene expression)

  • Culminate with physiological effects (muscle contraction, metabolic changes)

  • Connect to behavioral/organismal outcomes (development, reproduction)

• Multi-level Experimental Integration:

  • Molecular Level:

    • Receptor cloning and expression

    • Peptide-receptor interaction studies

    • Structure-activity relationship analyses

    • Signaling pathway identification

  • Cellular Level:

    • Calcium imaging in receptor-expressing cells

    • Phosphorylation cascades

    • Transcriptional responses

    • Cellular physiological outputs

  • Tissue Level:

    • Ex vivo tissue preparations

    • Organ-specific effects

    • Electrophysiological recordings

    • Contractility measurements

  • Organismal Level:

    • Developmental timing effects

    • Reproductive behaviors

    • Physiological state alterations

    • Comparative studies across species

• Technological Integration:

  • Combine mass spectrometry with electrophysiology

  • Link receptor pharmacology with behavioral assays

  • Connect transcriptomics with functional outputs

  • Integrate computational modeling with experimental validation

• Temporal Considerations:

  • Examine acute versus chronic effects

  • Study developmental stage-specific responses

  • Investigate circadian or seasonal variations in sensitivity

  • Evaluate evolutionary changes across taxonomic groups

• Data Integration Approaches:

  • Apply systems biology frameworks

  • Develop predictive models of peptide action

  • Create comprehensive databases of pyrokinin effects

  • Use machine learning to identify patterns across experimental datasets